Journal: Heliyon

Article Title: Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo

doi: 10.1016/j.heliyon.2024.e28021

Figure Lengend Snippet: Cap inhibits Pi-induced calcification of VSMCs by inhibiting the Wnt/β-catenin signaling pathway by upregulation of TRPV1 receptor expression. A) RT-qPCR showing the mRNA levels of Wnt3a and β-catenin; B) Western blot illustrating the protein expression levels of Wnt3a and β-catenin; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR showing the expression of osteogenesis-specific and phenotypic markers in VSMC calcification. F) Western blot depicting the protein expression levels of osteogenesis-specific and phenotypic markers in VSMC calcification; G) Western blot showing the protein expression of β-catenin after treatment with DKK1. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Pi + Cap + DKK1: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 20 ng/mL DKK1; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies against BMP-2 (PRP100126) and Runx2 (ABP53087) were purchased from Abbkine (Wuhan, Hubei, China); SM22α (AF9266) and Wnt3a (DF6113), Affinity Biosciences LTD; β-actin (81115-1-RR), proteintech (Hubei, China); β-catenin (ab32572), Abcom (USA); and DKK1 ( HY-P73305 ), MCE China (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Calcium Colorimetric Assay, Cell Culture, Control

Journal: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 3. Gene expression analysis of Wnt signalling in monolayer (Mono) and anoikis resistant (AR) cells of normal breast cell lines (N), ER-ve and ER+ve breast cancer cell lines. A) Protein expression of activated B-catenin (unphosphorylated), Lef1, Axin2, DKK1 and B-actin (housekeeper) in MCF7 monolayer and AR cells. B) Cluster analysis was performed using the fold change in expression

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Gene Expression, Expressing

Journal: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 4. Modulation of Wnt signalling in normal and breast cancer cell lines. Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) MCF10a cells (Wnt3a) B) MCF7 cells (Wnt3a) C) MDA-MB-231 cells (Wnt3a) D) MCF10a cells (DKK1) E) MCF7 cells (DKK1) F) MDA-MB-231 cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * ,0.01 ** ,0.001 generated by a T-test. G) Image of a MCF10a mammosphere H) Image of an MCF7 mammosphere I) Image of an MDA-MB-231 mammosphere. Scale bar represents 50 mM. doi:10.1371/journal.pone.0067811.g004

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Cell Culture, Control, Generated

Journal: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 5. Modulation of Wnt signalling in normal and primary breast cancer samples (Normal n = 3; ER+ve n = 3; ER-ve n = 3). Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) primary normal breast cells (Wnt3a) B) ER+ve primary breast cancer cells (Wnt3a) C) ER-ve primary breast cancer cells (Wnt3a) D) primary normal breast cells (DKK1) E) ER+ve primary breast cancer cells (DKK1) F) ER2ve primary breast cancer cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * .0.01 ** .0.001 generated by a T-test. G) Image of a normal primary mammosphere H) Image of an ER positive primary tumour mammosphere I) Image of an ER negative primary tumour mammosphere. Scale bar represents 50 mM. doi:10.1371/journal.pone.0067811.g005

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Cell Culture, Control, Generated

Journal: Oncotarget

Article Title: Positive feedback loop of hepatoma-derived growth factor and β-catenin promotes carcinogenesis of colorectal cancer

doi:

Figure Lengend Snippet: A. - B. β-catenin knockdown significantly suppressed HDGF mRNA expression in HCT116 A. and HT29 B. cells by real-time PCR analysis, respectively; C. - E. β-catenin knockdown inhibited HDGF protein expression in HCT116 cells C. and mainly inhibited nuclear HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 D. and HT29 E. cells; F. Recombinant Wnt3a and DKK1 increased and decreased HDGF and β-catenin expression in HCT116 cells, respectively; G. - I. Recombinant Wnt3a and DKK1 increased and decreased nuclear and cytoplasmic HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 G. , I. and LOVO H. cells by Western blot analysis, respectively.

Article Snippet: To further verify the effect of β-catenin on HDGF expression in CRC cells, HDGF and β-catenin protein expressions in HCT116 were induced by 100ng/ml human recombinant Wnt3a (R&D SYSTEMS) and inhibited by 200ng/ml human recombinant DKK1 (R&D SYSTEMS) for 48 hours by Western blot analysis, respectively (Figure ).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 1. In vitro evaluation of the proper effect of the BHQ880 antibody over Dkk1 protein neutralization. (A) Representative images of B1a/7TGC cell reporter line activation when stimulated by the GSK-3 inhibitor CHIR99021 (positive control) or by increasing doses of recombinant mouse Wnt3a (mWnt3a), and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. Non-treated (NT); # vs. 0 ng/mL mWnt3a. (B) Representative images of B1a/7TGC cell reporter line inactivation when previously stimulated cells with 150 ng/mL of mWnt3a were turned off through the addition of increasing doses of recombinant rDkk1, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; # vs. 0 ng/mL rDkk1. (C) Representative images of the BHQ880 neutralizing effect over rDkk1 protein in the B1a/7TGC reporter cells stimulated with 150 ng/mL of mWnt3a, together with 250 ng/mL of rDkk1 and increasing doses of BHQ880 antibody, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; & vs. Wnt3a; # vs 0 ng/mL BHQ880. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between groups. In all cases, scale bars = 50 μm and the data are presented as the mean + SEM. **p <0.01; ***p <0.001; #p <0.05; ##p <0.01; ###p <0.001; &&p <0.01.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: In Vitro, Neutralization, Activation Assay, Positive Control, Recombinant

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 2. Protein expression of Dkk1 in the healthy and injured spinal cord. The figure shows representative images from the immunohistochemical evaluation of Dickkopf-1 (Dkk1) protein expression in the non-lesioned (NL) (n = 5) spinal cord (A) and after SCI at 24 hours post-injury (hpi) (n = 4), and 3 days post-injury (dpi), 7 dpi (n = 4), 14 dpi (n = 5), 28 dpi (n = 3), and 42 dpi (n = 5) at the lesion epicenter (B-G). Dashed line squares in A-G indicate the areas shown in the corresponding higher magnification images. Scale bars = 500 μm. Inserts scale bars = 50 μm. (H) Densitometric analysis of Dkk1 immunolabeling in the NL spinal cord and at the injury epicenter after SCI from 24 hpi to 42 dpi. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between NL and the different times post-injury. In all cases, the data are presented as the mean + SEM. **p <0.01; ***p <0.001.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Expressing, Immunohistochemical staining, Immunolabeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 3. Analysis of the circulating levels of Dkk1 in Serum samples from non- lesioned (NL) and lesioned rats at 24 hours after damage (SCI). The figure shows data obtained from the quantitative analysis of serum circulating Dkk1 protein levels measured by ELISA in NL animals (n = 9) and injured animals 24 hours after SCI (n = 9). No significant differences were observed between groups. A two-tailed t test was used to determine the existence of significant differences between groups. The data represent the mean ± SEM.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Serum-starved K562 cells were treated with PBS (control) or Wnt3a (100 ng/ml) or Wnt3a + Dickkopf peptide (DKK1, 200 ng/ml) for 24 hr prior to EV isolation. Biochemical analysis of cellular (Cells) and EV fractions was performed by immunoblotting of extracts using antibodies against antigens shown. Detection of ß-Catenin was carried out using cytosolic fractions. ( B ) Quantitation of EV marker protein levels from EV immunoblots shown in ( A ). ( C ), Serum-starved K562 cells were treated with DMSO (control) or CHIR99021 (10 µM) for 24 hr prior to EV isolation. Biochemical analysis of EV fractions was performed as in ( A ). Representative immunoblots for a control experiment along with two replicate CHIR99021-treated samples are shown. ( D ) Quantitation of protein levels from EV immunoblots in ( C ). ( E ) Quantitation of LAMP1 protein from cellular fractions in ( C ). Molecular mass marker mobility is shown at the left of immunoblot panels in kilodaltons. t test: *p<0.05; **p<0.01; error bars represent SEM; n ≥ 3. ( F ) Flow cytometry determination of LAMP1 levels in anti-LAMP antibody-labeled control and CHIR99021-treated cells. Confocal images at right show lysosomes stained with anti-LAMP1 antibody (green) and nuclei labeled with DAPI (blue). Bar,10 µm.

Article Snippet: The next day, the medium was replaced with fresh EV-free RPMI supplemented with 100 ng/ml recombinant Wnt3a protein (R and D Systems), or Wnt3a combined with 200 ng/ml recombinant DKK1 protein (R and D Systems) and EVs were collected 24 hr later.

Techniques: Control, Isolation, Western Blot, Quantitation Assay, Marker, Flow Cytometry, Labeling, Staining

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A Two epitope bins are apparent amongst the Twist anti-DKK1 leads from this epitope binning analysis. The formation of Antibody-Antigen-Antibody complexes indicates the antibodies are not binding to the same epitope of DKK1. B Anti-DKK1 lead antibodies bind to hDKK1 cysteine-rich domain CRD1 or CRD2 or both CRD1 and CDR2 (in the instance of bispecific antibodies), and cross-reactivity with mouse and cynomolgus monkey DKK1. The assays were repeated in triplicate.

Article Snippet: On Day 2, mWNT3a (R&D Systems, #1324-WN, 50 ng/mL) and hDKK1 (R&D Systems, #5439-DK/CF, 500 ng/mL) were incubated with anti-DKK1 antibodies in assay buffer (BPS Bioscience Thaw medium 1, #60187 with 10 mM LiCl) at 37 °C for 30 min.

Techniques: Binding Assay

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A Wnt TCF/LEF reporter assay screening. Wnt TCF/LEF signaling is blocked by DKK1 binding to LRP5/6. Anti-DKK1 antibodies that bind to hDKK1 CRD2 block the binding of DKK1 to the co-receptors, and lead to the reactivation of Wnt canonical signaling. B MC3T3.E1 cell differentiation detection by mineralization assay. Soluble hDKK1 suppresses pre-osteoblast cell differentiation via the Wnt canonical pathway. Anti-DKK1 antibodies that bind to hDKK1 CRD2 block the binding of DKK1 to the LRP5/6 co-receptors and restore cell differentiation. C Wnt non-canonical phospho-JNK detection. Colo205 cells were treated with Wnt, DKK1, and anti-DKK1 lead antibodies. Intracellular JNK phosphorylation level was detected with ELISA. D Primary immune cell activation. DKK1 leads to immune suppression including T cell inactivation, MDSC accumulation, and NK cell clearance. GM-CSF is the biomarker for NK cell activation. Human PBMC were treated with an immune stimulator, mWNT3a, hDKK1, and DKK1 lead antibodies. Cytokine release of GM-CSF was measured by ELISA. Antibodies binding to CRD1 of DKK1 showed stronger NK cell activation. E PC3 tumor cell cytotoxicity by activated immune cells. T cells and NK cells in human PBMC were activated and co-cultured with PC3 tumor cells for 6 days. Activated immune cells kill PC3 cells, while hDKK1 treatment inhibits cytotoxicity. Blocking the interaction of hDKK1 to the receptor with Twist DKK1 lead antibodies restores the cytotoxicity potency. Antibodies binding to CRD1 of DKK1 showed stronger cytotoxicity. F Anti-DKK1 antibody targeting DKK1 CRD1 also induced cytotoxicity in breast, gastric and colon cancer cells. Using the KILR cytotoxicity assay, a high luminescence signal was detected in cytotoxic cells. All the samples were duplicated, and the assays were repeated three times.

Article Snippet: On Day 2, mWNT3a (R&D Systems, #1324-WN, 50 ng/mL) and hDKK1 (R&D Systems, #5439-DK/CF, 500 ng/mL) were incubated with anti-DKK1 antibodies in assay buffer (BPS Bioscience Thaw medium 1, #60187 with 10 mM LiCl) at 37 °C for 30 min.

Techniques: Reporter Assay, Binding Assay, Blocking Assay, Cell Differentiation, Mineralization Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Activation Assay, Biomarker Discovery, Cell Culture, Cytotoxicity Assay

Journal: Journal of personalized medicine

Article Title: Impact of REAC Regenerative Endogenous Bioelectrical Cell Reprogramming on MCF7 Breast Cancer Cells.

doi: 10.3390/jpm13061019

Figure Lengend Snippet: Figure 7. DKK1 and SFRP1 quantification by ELISA. The concentrations of DKK1 (A) and SFRP1 (B) were measured after 7, 10, and 14 days in supernatants of MCF7 either exposed or not to REAC TO-RGN treatment. Data are expressed as mean ± SD relative to the control (mean ± SD) (** p ≤0.01; *** p ≤0.001).

Article Snippet: The concentrations of human secreted frizzled-related protein 1 (SFRP1) and human Dickkopf-related protein 1 (DKK1) were determined using the human secreted frizzledrelated protein 1 (SFRP1) ELISA Kit (Cusabio, Flarebio Biotech LLC) and the human DKK1 (Dickkopf-related protein 1) ELISA Kit (Elabscience Biotechnology Co., Ltd, Houston, TX, USA), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Nuclear β-catenin in vessels of human astrocytoma does not correlate with WHO grade. (A) TCGA database analyses for Wnt1 , Wnt3a , Wnt5a , Wnt7a , Wnt7b , Dkk1 , and Dkk2 mRNA expression as log2-fold expression. Differences in mRNA expression in GBM compared with normal central nervous system tissue (dashed red line) are shown. (B and C) Paraffin sections of three normal human brains (B) and five different human astrocytoma WHO grades I–IV (C) stained for β-catenin and analyzed for its endothelial, nuclear localization. Insets show individual nuclei in higher magnification. Bars: (B) 14 µm; (C) 20 µm.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Wnt1 expression decreased subcutaneous GL261 tumor growth and increased animal survival. (A) Western blots showing Wnt1 and Dkk1 expression +/−DOX in GL261 cells. (B) sTOP-FLASH assay on human embryonic kidney (HEK293)/GL261 co-cultures without DOX. Monoculture of transfected HEK293 cells served as baseline. Wnt1 and Dkk1 cells were cultured without (−) or with (+) Wnt3aCM (one experiment in triplicate). (C) In vitro proliferation of the control-, Wnt1-, and Dkk1-GL261 line cultured +/−DOX. (D, left) Representative pictures of NUDE mice with subcutaneous tumors −DOX. (right) Tumor volumes ( n = 7/group) of the transplanted glioma cell lines −DOX (*, P < 0.05; **, P < 0.01). (E) H&E-stained paraffin sections revealed reduced necrotic areas for Dkk1-expressing tumors (N). (F, left) Pimonidazole (brown) immunohistochemistry staining revealed tumor hypoxia, hematoxylin counterstaining (blue). (right) Hypoxia in Dkk1 −D compared with the control −D (*, P < 0.05) and Wnt1 −D tumors (**, P < 0.01; n = 4 tumors/group, slices from the center of similar sized tumors). (G, left) Representative vessels, stained for nuclear β-catenin (brown) and hematoxylin (blue) of control −D , Wnt1 −D , and Dkk1 −D tumors. Arrowheads point to β-catenin + nuclei. (right) Quantification of β-catenin + nuclei ( n = 4 tumors/group, 20 vessels/tumor; ***, P < 0.001). Bars: (D) 1 cm; (E and F) 400 µm; (G) 35 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Expressing, Western Blot, Transfection, Cell Culture, In Vitro, Control, Staining, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 reduced and normalized subcutaneous tumor vascularization, whereas Dkk1 caused the opposite effect. (A) Tumor growth in Wnt1 −D compared with Wnt1 +D condition (left; n = 12/group; **, P < 0.01; ***, P < 0.001) and mouse survival (right; n = 12/group). (B) Tumor volume for Dkk1 −D compared with Dkk1 +D condition (left; n = 12/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001) and mouse survival of Dkk1 +/−D tumors (right; n = 12/group). (C, top) IF staining on subcutaneous Wnt1 tumors for CD31/PECAM-1, α-SMA, and TOPRO-3. (bottom left) Vessel density of subcutaneous Wnt1 tumors +/−DOX ( n = 5 tumors/group, 10 pictures/tumor; **, P < 0.01). (bottom right) Association of α-SMA + cells to ECs in Wnt1 +/−D tumors ( n = 5 tumors/group, 10 vessels/tumor; ***, P < 0.001). (D) Same staining and experimental settings as in C for subcutaneous Dkk1 +/−D tumors (*, P < 0.05). (C and D) Insets show vessels in higher magnification. (E) Perfusion with isolectin revealed that vessels from control −D , Wnt1 −D , and Dkk1 −D tumors exhibited blood flow. Bars: (C and D) 400 µm; (E) 200 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Glioma-derived Wnt1 up-regulated endothelial Dll4 , leading to a stalk cell–like gene signature. (A) ISH against Dll4 (black) and hematoxylin (blue) of subcutaneous control −D , Wnt1 −D , and Dkk1 −D GL261 tumors, showing representative small (0–300 µm 2 ; left) and large (>300 µm 2 ; right) vessels. (B, left) Quantification of vessel diameter ( n = 3 tumors/group, 15 pictures/tumor) grouped in categories of 0–30 µm 2 , 31–300 µm 2 , and >300 µm 2 (*, P < 0.05; **, P < 0.01). (right) Quantification of Dll4 + ECs ( n = 6 tumors/group, 5 small and 5 large vessels/tumor; ***, P < 0.001). (C, top) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells (asterisks indicate HUVECs). (bottom) qRT-PCR ( n = 6) for human genes regulated by Wnt1 −D and Dkk1 −D compared with control −D co-cultures (red line). (D) qRT-PCR for mouse genes regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (red line; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars: (A) 50 µm; (C) 80 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Control, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor cell–free Matrigel plug assay confirmed the antiangiogenic effect observed in Wnt1-GL261 tumors. (A) Immunohistochemistry staining for CD31/PECAM-1 (red) and hematoxylin counterstaining (blue) of Matrigel plug sections, supplemented with diluent (control), 400 µg/ml human Wnt1, or 200 µg/ml mouse Dkk1 recombinant proteins. Bar, 200 µm. (B) Quantification of CD31 + area in percentage in Matrigel plugs ( n = 6 plugs/group, 8 representative pictures/plug; **, P < 0.001). Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Matrigel Assay, Immunohistochemistry, Staining, Control, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 normalized vascularization and retained vascular barrier properties in GL261 glioma. (A, left) Representative H&E staining of serial thick sections of tumor-bearing mouse brains. (right) Corresponding IF staining for Podxl and TOPRO-3. (B, top) Quantification of glioma volume based on H&E staining (control −D n = 3, Wnt1 −D n = 6, and Dkk1 −D n = 4; *, P < 0.05). (bottom) Podxl + vessel areas within tumors (control n = 4, Wnt1 n = 6, Dkk1 n = 5; **, P < 0.01). (C) IF staining for CD31/PECAM-1 and desmin ( n = 3 tumors/group, 8 pictures/tumor; *, P < 0.05). (D) Large image reconstructions of IF staining for mouse endogenous IgG (mIgG), Podxl, and TOPRO-3 on brain sections bearing control −D , Wnt1 −D , and Dkk1 −D glioma. Bars: (A) 200 µm; (C) 50 µm; (D, top) 1 mm; (D, bottom) 27 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Pdgfb is a Notch-independent target of β-catenin signaling in ECs. (A) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells. qRT-PCR ( n = 4) for human PDGFB regulated by Wnt1 −D or Dkk1 −D (ns) compared with control −D (gray line; *, P < 0.05). (B) qRT-PCR for Pdgfb gene regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (gray line; n = 4; *, P < 0.05; ***, P < 0.001). (C, left) qRT-PCR for Axin2 and Pdgfb regulated in β-catenin–deficient ECs transduced with LefΔN-βCTA compared with the vector control (gray line; n = 4; *, P < 0.05; **, P < 0.01). (right) Representative Western blot ( n = 1) for PDGF-B from LefΔN-βCTA ECs compared with the vector control; top band shows the nonreduced (PDGF-BB) and bottom band reduced protein (PDGF-B). Densitometric analysis shows the summarized values for both forms of PDGF-B of samples loaded twice. (D) qRT-PCR of β-catenin–deficient ECs (control) or transduced with LefΔN-βCTA grown for 24 h either on gelatin- or Dll4-coated dishes ( n = 3). Notch pathway induction was monitored by Hes1 expression (*, P < 0.05; **, P < 0.01). Notch signaling was blocked by Dll4-Fc. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Control, Quantitative RT-PCR, Transduction, Plasmid Preparation, Western Blot, Expressing